The capability to regenerate lost or damaged organs is severely limited in humans and most vertebrate animals. Other animals, in contrast, have remarkable regenerating abilities. Among these are the echinoderms, a group of animals closely related to vertebrates. We have been using the sea cucumber Holothuria glaberrima as a model system to study intestinal regeneration. These animals can regenerate their complete digestive tract following a process of auto-evisceration. Our laboratory has characterized the intestinal regeneration process and the associated cellular events. In recent years we have begun to identify genes that might control different aspects of the regeneration process by using molecular tools, particularly the construction and analysis of an EST database and the use of microarrays. These experiments have led to the identification of candidate genes differentially expressed during intestinal regeneration. The present proposal focuses on 20 of these genes. We propose to use these candidate genes to study the molecular control of intestinal regeneration. We intend to validate their differential expression and characterize their sequence, spatial and temporal expression patterns, using in silico methods as well as QRT-PCR, Western blotting, in situ hybridization and immunohistochemistry. This will lead to determining the cellular or tissue component where they are being expressed and a putative function for the genes. More importantly, we will determine the gene's function by knock-down experiments using interference RNA (RNAi) in conjunction with various markers that serve to determine the cellular processes that occur during regeneration such as: changes in tissue cellular components and morphology, formation of the regeneration blastema, intestinal lumen, enteric nervous system, and tissue innervation, as well as changes in cell populations, migration, division, death, dedifferentiation, and growth among others. Our results will help discover novel genes associated with intestinal regeneration and characterize their expression to particular cells and regenerative events. Moreover, their function will be determined, and mechanisms of how they might control the intestinal regeneration process will be identified. We expect that our experiments will make available information, that can be used to study the limitations of vertebrate regenerative capacities and that will serve as the basis for the development of new therapeutics/drugs that modulate regenerative processes.